In Vitro Assessment of the Toxic Effects of an AKWATON based-disinfectant onHuman TissuesMathias K Oulé*, Christine Lesage, Julie Gauvin, Mona Friesen, Michael Dickman, Anne-Marie Bernier and Lamine Diop
Department of Experimental Science, Faculty of Science, Université de Saint-Boniface, Canada
*Corresponding author: Mathias K Oulé, Department of Experimental Science, Faculty of Science, Université de Saint-Boniface, 200, de la Cathédrale Avenue,Winnipeg, MB, R2H 0H7, Canada, Tel: 204 237 1818 (Extn: 499); E-mail: [email protected]
Received date: April 11, 2017; Accepted date: April 26, 2017; Published date: May 04, 2017
The purpose of this study is to prove the potential safe use of AKWATON as a new antimicrobial product. Manyservice products are often removed from the market due to their toxic effects on the human body or to theiraggressiveness towards the environment. Antimicrobial products such as disinfectants may contain harmfulingredients that can cause disease. Some disinfecting products are corrosive or irritating; others produce strongodors, which in the long run can cause real health problems. AKWATON is a new disinfectant, member of the familyof guanidine polymers.
Its bactericidal, fungicidal and sporicidal properties have been demonstrated and widely documented. In thisstudy, the toxic effects of AKWATON and of three well known commercial antimicrobial products currently on market,were evaluated and compared on various human tissues including eyes, lung, skin and liver cells. The testing wereperformed using the TB (Trypan blue) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)methods. Cell-cultures and the different tests done, showed that the AKWATON based-disinfectant was much lesstoxic, killing many fewer cells than the commercial disinfectants. It spared more than 64% of skin cells; 65% of lungcells; 66% of eye cells and 64% of liver cells while some well-known disinfectants currently marketed killed 100% ofcells. This study demonstrated that AKWATON can be used as an odorless, colorless, non-corrosive and harmlessdisinfectant for hospital, agriculture industry, farming, food service and household facilities or as antiseptic.
IntroductionDisinfection ensures the partial or total removal of microorganisms
on soiled objects (instruments, clothing, surfaces, etc.) while anychemical treatment applied to living tissue in order to destroy oreliminate potentially pathogenic microorganisms, or slow theirgrowth, is called antisepsis. Chemicals used to clean objects andsurfaces in contact with microorganisms are called disinfectants andthose applied to living tissues are called antiseptics [1]. The history ofthe use of chemicals to fight micro-organisms goes back to the time ofancient Egypt [2]. In the 19th century, disinfection and antisepsischanged with the discovery of chemicals such as chlorine by CarlScheele; iodine by Bernard Courtois and hydrogen peroxide by LouisThénard [3]. Today disinfection and antisepsis are daily acts applied ina multitude of environments (households, industries, hospitals, publicplaces, etc.). Antimicrobial chemicals are used every day by millions ofpeople around the world. Unfortunately, they do not have selectiveaction and affect both microorganisms and mammalian cells [4].Disinfectants and antiseptics contain active molecules that, not only,inhibit the growth of microorganisms or kill them, but also producetoxic effects on human and animal cells [5,6]. On the other hand,active ingredients in some antimicrobial products induce resistance,even to antibiotics in bacteria [1]. This is the case of triclosan [7,8]. Forthis reason, governments increasingly remove certain service productsfrom the market. Recently in USA, The FDA (Food and DrugAdministration) banned the use of triclosan, triclocarban and 17 other
chemicals in disinfectants for hands and in liquid soaps [9,10].Furthermore like triclosan, many other chemical ingredients areassociated with allergies [11,12]. To avoid the toxic effects of thechemicals we use every day, and to preserve the environment, it istherefore necessary to explore other molecules capable of killingmicroorganisms without affecting human and animal organisms.
Antimicrobial biocides have long been in use in domestic andclinical environments. For over half a century, cationic biocides havebeen prominent among other agents used to combat cross-infectionsand have contributed to the overall reduction in nosocomial infections[13]. Correct application of these biocides plays a very effective role inthe elimination of infection in veterinary, dental, domestic and hospitalsettings [1]. Polyhexamethylene biguanide (PHMB), a member of thepolymeric guanidine family, has broad-spectrum activity againstGram-positive and Gram-negative bacteria, fungi, yeasts [14] andviruses, including human immunodeficiency virus [15]. It has beenwidely used for many years as an antiseptic in medicine and the foodindustry, as a mouthwash [16], as a disinfectant for a variety of solidsurfaces [17] and also in water treatment [18]. AKWATON is apolyhexamethylene guanidine hydrochloride (PHMGH) basedformulation of a novel disinfectant. PHMGH, a member of theguanidine family, is a polymer with bactericidal, fungicidal, andsporicidal, properties that has been demonstrated to work at lowconcentrations with short contact times [19-21]. The bactericidalactivity against E. coli and meticillin-resistant Staphylococcus aureus(MRSA) and the mode of action of PHMGH have been clearlydemonstrated by Oulé et al. [20]. Their results showed that no matterwhat type of water (distilled, tap and hard water) was used to make
Oulé et al., J Antimicrob Agents 2017, 3:2DOI: 10.4172/2472-1212.1000140
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dilutions, AKWATON killed MRSA and E. coli respectively at a lowconcentration of 0.04% and 0.005% within 1.5 minutes [19]. Theseauthors also demonstrated that the minimum sporostaticconcentration, the minimum sporicidal concentration and the timerequired for sporicidal activity corresponded to 0.06% (w/v), 0.08%(w/v) and 8.5 minutes, respectively [19]; and that the minimuminhibitory concentration, the minimum fungicidal concentration(MFC) and time required for the fungicidal activity of AKWATON atthe MFC were 0.025% (w/v), 0.045% (w/v) and 2.5 min, respectivelyOulé et al. [20].
Other previous studies have demonstrated that PHMGH, the activeingredient of the new AKWATON-based disinfectant, is an odourless,colourless and noncorrosive polymer with high solubility in water [22]and is significantly less toxic and harmless than currently useddisinfectants [14,23] to humans and animals at a concentration ≤ 1%.In addition, cell cultures and the different tests carried out by Oulé etal. [20], showed that the new AKWATON-based disinfectant killedfewer animal cells than the commercial disinfectants, sparing 80% ofrat pancreatic (C2C12) cells and 65% of muscle RnM5F cells, whilstsome of the well-known disinfectants currently on the market killed85-100% of those cells.
To demonstrate its potential use as a less hazardous antibacterialthan the currently used disinfectants, and to validate its use as anantiseptic and an ideal disinfectant for households and hospitals, thetoxic effects of AKWATION have been assessed on human tissues.
Material and MethodsHuman cells used in this study were lung cells (IMR-90 (ATCC®
CRL-2302™)), purchased from American Type Culture Collection(ATCC) ((Manassas, Va., USA). All the reagents have been purchasedfrom Sigma Chemical Co. (St Louis, MO, USA). Cells were maintainedat 37°C under a continuous 5% CO2 atmosphere.
Assessment of the effect of disinfectants on cellsAKWATON (0.05%) and three well-known and currently marketed
antimicrobial chemicals including Ethanol (70%), DEXIDIN-4, and thecommercial disinfectant LYS purchased from Canadian Real SuperStore (Winnipeg, MB, Canada) were used to treat the four types ofhuman cells. Skin cell (A-431 (ATCC® CRL-1555™)) cells and eye cells(ARPE-19 (ATCC® CRL-2302™)) were plated at 7,500 cells/cm2 andcultured in DMEM (Dulbecco’s Modified Eagle’s Medium); and Lungcells (IMR-90 (ATCC® CCL-186™)) and liver cells (Hep G2 [HEPG2](ATCC® HB-8065™)) in EMEM (Eagle's Minimum Essential Medium).
All media were supplemented with 1% Penicillin, 1% Streptomycin,2 mM glutamine and 10% fetal bovine serum (FBS). Cells wereincubated at 37°C under a continuous 5% CO2 atmosphere for 24 hourto 65-75% confluency. Before each test, cells were detached using0.05% trypsin. Cells were exposed to AKWATON-based disinfectantand to three other well-known commercial antimicrobial chemicals for10 minutes.
Cultures with ≥ 95% viable cells were used for experiments. Theviability of cells before and after treatment was tested by trypan blueexclusion tests and MTT test. Cells were observed under a microscopeand counted using a hemocytometer. Calculation: Cell
Statistical analysisThree different batches of the AKWATON-based disinfectant and
four three well-known commercial antimicrobial chemicals were usedto treat the four types of cells. For AKWATON, reported data was theaverage of the results from the three batches tested. Each test with eachchemical was performed in duplicate and repeated three times. Resultswere analyzed using one-way analysis of variance and Student’s t-test.Differences with a value of P<0.05 were considered statisticallysignificant.
DiscussionThe purpose of applying disinfectants and antiseptics is to kill
microorganisms or prevent their development. But an equallyimportant thing is to ensure that the application of these antimicrobialproducts does not affect the human and animal health and does notaffect the environment, which is a real concern with the majority ofcommercial chemicals currently on the market. The advent of newbacterial strains that are increasingly resistant to antibiotics anddisinfectants is a serious problem that deserves more attention and towhich a durable solution must be found.
This is why it is necessary to develop new molecules with powerfulantimicrobial properties, but harmless for human and animalorganisms and for the environment. AKWATON is a new colorless,odorless, non-corrosive antimicrobial product with excellentmicrobicidal power. Its antimicrobial activities have been widelydocumented [19,25-27].
Oulé et al. [19] have clearly demonstrated its bactericidal activityagainst Staphylococcus aureus, Pseudomonas aeruginosa, Salmonellacholeraesuis, meticillin-resistant S. aureus (MRSA) and E. coli; itssporicidal activity against Bacillus subtilis spores [20] and its fungicidalactivity against Trichophyton mentagrophytes [4]. In addition to itsmicrobicidal properties, it has been demonstrated that AKWATONwas much less toxic to rat cells than well-known disinfectants currentlyon the market. Oulé et al. [4] established that the AKWATON-baseddisinfectant was much less toxic to rat pancreatic and muscle cells.
The AKWATON-based disinfectant killed fewer cells than thecommercial disinfectants, sparing 80% of C2C12 (ATCC® CRL-1772™)cells and 65% of RIN-m5F (ATCC® CRL-11605™) cells, whilst LYS andMCL, currently on the market, killed 85%-100%. The purpose of thisin vitro study is to show that AKWATON is also less toxic to humancells than commercial antimicrobial products. The in vitro tests withcell lines are a good alternative to the use of laboratory animals intoxicological studies. Animal protection organizations are increasinglyopposed to laboratory experiments on animals, because of the crueltyinherent in these practices [28].
In this study, the toxic effects of AKWATON (0.05%) (a novelantimicrobial product), Ethanol (70%), DEXIDIN-4 (a commercialantiseptic) and LYS (a commercial disinfectant) were assessed onhuman cells including eyes, skin, lung and liver cells in In-vitroexperiments. Figure 1 shows untreated cells which, have served as acontrol to observe and evaluate the effects of the four antimicrobialproducts tested. Untreated cells were numerous, healthy and evenlydistributed in the visual field of the microscope.
Citation: Oulé MK, Lesage C, Gauvin J, Friesen M, Dickman M, et al. (2017) In Vitro Assessment of the Toxic Effects of an AKWATON based-disinfectant on Human Tissues. J Antimicrob Agents 3: 140. doi:10.4172/2472-1212.1000140
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The Figure 2 compares the survival of the four types of human cellsexposed to various chemicals mentioned above. Each antimicrobialproduct killed the same percentage of cells in the four cell types. Foreach antimicrobial product, there was no significant differencebetween the cell types (P>0.05). However, for each cell type asignificant difference was observed between antimicrobial products(P<0.05), except between AKWATON (0.05%) and Ethanol (70%)(P>0.05). Among the four antimicrobial products, LYS was highlytoxic, killing 100% of each of the four cell types used, probably due toits chemical composition.
Figure 2: Viability of 4 human cell types (Skin, Lung, Eyes and Livercells) after treatment with different antimicrobial products. Controlcells were untreated.
The commercial disinfectant LYS used in this study is a mixture ofseveral active ingredients such as Ethyl alcohol, Butane, Propane andN-Alkyl-dimethyl benzyl ammonium chloride. The morphology ofcells after their exposure to LYS suggests that the disinfectant attackedvarious targets on cells. Their appearance suggests that some activemolecules in the disinfectant LYS fragmented the plasma membranefollowed by penetration of other actives molecules into the cells thatattack cytoplasmic components, causing loss of the cytoplasm, totalcollapse of the cells and resulting in an appearance of puree of the cellmass. Figure 3 shows cells after a 10 min-treatment with LYS. All cellswere completely destroyed.
Figure 3: Effects of the commercial disinfectant LYS on human cells;(A): Skin cells; (B): Lung cells; (C): Eyes cells and (D): Liver cells.
DEXIDIN-4 also caused a high percentage of cell death killingapproximately 75% of cells in each cell type. Figure 4 shows cells aftertheir treatment with DEXIDIN-4. DEXIDIN-4 was toxic to cells butless than LYS. There was a significant difference between DEXIDIN-4and LYS (P<0.05). DEXIDIN-4 is used as an antimicrobial surgicalcleaner; antiseptic cleaner for skin and minor injuries and for handwashing in hospitals, labs and food services. DEXIDIN-4 is composedof 4% chlorhexidine gluconate and 4% isopropyl alcohol. Dependingon the concentration, each of these two chemical compounds can betoxic to mammalian cells, and their modes of action on cells are verywell documented. Isopropanol is an active ingredient that can causecell death at relatively low concentrations such as 4% in the antisepticDEXIDIN-4. Kasajima, et al. [29] reported in an in vitro study that theinhibition of DNA, RNA and protein-syntheses of the mammalian cellswas induced by treatment with ethyl alcohol at 1% to 10% (V/V) for 2hours, in a dose-related manner. In DEXIDIN-4, the other main activeingredient is 4% chlorhexidine gluconate. An in vitro study byFlemingson et al. [30], comparing the effects of three mouth rinses onhuman gingival fibroblasts, showed that these three rinses were toxic tothe cells, chlorhexidine being the most cytotoxic. Taner et al. [31] havealso demonstrated the genotoxic and cytotoxic effects of chlorhexidineon human lymphocytes. According to Nancy and Don [32], culturedhuman periodontal cells treated with 2% chlorhexidine exhibited afoamy appearance in which most of the cytoplasm seemed to havebeen extracted from the cells. According to an in vivo genotoxicity
Citation: Oulé MK, Lesage C, Gauvin J, Friesen M, Dickman M, et al. (2017) In Vitro Assessment of the Toxic Effects of an AKWATON based-disinfectant on Human Tissues. J Antimicrob Agents 3: 140. doi:10.4172/2472-1212.1000140
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study conducted by Grassi et al. [33] in rats, chlorhexidine inducedDNA damage in leukocytes, renal cells and oral mucosal cells. And inan in vitro study on rat oral mucosal cells and rat leukocytes, Ribeiro etal. [34] demonstrated that Chlorhexidine was highly cytotoxic,inducing oxidative stress and apoptotic and necrotic cell death. In thisstudy, the treatment with DEXIDIN-4 resulted in the death ofapproximately 75% of cells of each cell type. Such high rate of celldeath results directly from the elevated toxicity of DEXIDIN-4 on thecells. This high toxicity could be attributed to the combined actions ofalcohol and chlorhexidine on the cells.
Figure 4: Effects of Dexidin-4 on human cells; (A): Skin cells; (B):Lung cells; (C): Eyes cells and (D): Liver cells.
The other two antimicrobial products, AKWATON (0.05%) andEthanol (70%) produced less cytotoxic effects on each cell type. Whenthe cells were exposed to AKWATON (0.05%), respectively 64%, 65%,66% and 64% of the skin, lung, eye and liver cells survived (Figure 1).Similar results were observed with Ethanol (70%). There was nosignificant difference between their effects on the cells (P>0.05). Thesetwo chemicals (Ethanol and AKWATON) killed about 30% to 35% ofcells. Figures 5 and 6 show the appearance of the four cell types aftertheir treatment with AKWATON-0.05% and Ethanol (70%).
In an in vitro study, Lingna et al. [35] examined the effects ofEthanol on mouse skin cells and they observed a dose-dependent toxiceffect. When the concentration reached 50%, more than 75% of cellswere killed after 2 days of exposure. In this study, we observed about30% of cell death, probably because of the short exposure time of 10min. The cells that survived treatment with AKWATON-0.05% (Figure5) or with Ethanol (70%) (Figure 6) seemed to be healthy compared tountreated cells (Figure 2). The effect of Ethanol is known; it acts on thebiological membrane by interdigitating the two lipid layers, thusreducing its thickness and increasing its permeability [36]. Oulé et al.[19] suggested that the main target of PHMGH, the active molecule inAKWATON, seems to be the cell envelope. PHMGH would penetratethe cell envelope, attacking the bacterial cell wall and the membrane atthe same time.
Figure 5: Effects of AKWATON (0.05%) on human cells; (A): Skincells; (B): Lung cells; (C): Eyes cells and (D): Liver cells.
Figure 6: Effects of Ethanol-70% on human cells; (A): Skin cells; (B):Lung cells; (C): Eyes cells and (D): Liver cells.
The relative resistance of animal and human cells to AKWATONand Ethanol could be explained by the composition and structure oftheir membrane. For example, the presence of cholesterol and thequality of membrane lipids (length, saturation and level of branching)would enhance the integrity and the stability of the membrane ofanimal and human cells. Cholesterol is a major component ofmammalian cell membranes. It contributes to the stability and themaintenance of membrane structure by intercalating between the
Citation: Oulé MK, Lesage C, Gauvin J, Friesen M, Dickman M, et al. (2017) In Vitro Assessment of the Toxic Effects of an AKWATON based-disinfectant on Human Tissues. J Antimicrob Agents 3: 140. doi:10.4172/2472-1212.1000140
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phospholipids, acting as a pillar of resistance to attacks on themembrane. Essentially, cholesterol intervenes in the integrity, thestability and the maintenance of membrane fluidity [37]. Cholesterolalso contributes to the tightness of cell membranes, reducing theirpermeability to various substances [38,39]. The presence of cholesterolwould reduce the penetration of PHMGH and Ethanol into cells,which would mitigate their toxicity on cells.
One wonders why the presence of cholesterol in the cell membranecould not allow the cells to resist to LYS or DEXIDIN-4. In fact, thechemical composition of LYS and DEXIDIN-4 is complex. These twoantimicrobial products contain more than one active ingredient. LYSand DEXIDIN-4 are mixtures of several active components and werefound to be highly toxic to the four cell types (Figure 1). The presenceof several active molecules in an antimicrobial product can be effectivein eliminating or reducing the spread of potentially pathogenicmicroorganisms, but can also generate toxic effects on mammaliancells. In AKWATON there is only one active molecule, the PHMGH,which acts on cell membranes [19]. In this study, AKWATON wasshown to be much less toxic to human cells. In a previous study, Ouleet al. [4] showed that AKWATON (0.05%) was much less toxic to ratcells than commercial disinfectants. In another study, the same authorsshowed that AKWATON (0.04%) was very toxic for bacterial cells [19].It is known that the membrane of the bacterial cell does not containcholesterol. The presence of cholesterol would seem to be adetermining factor for the effect of AKWATON.
ConclusionThe growing resistance of pathogenic microorganisms to antibiotics
and antimicrobial chemicals, as well as the toxicity of these products tohuman and animal organisms and their aggressiveness to theenvironment, are real concerns. AKWATON, an odourless, colourless,stainless and non-corrosive substance with high bactericidal,fungicidal and sporicidal potency, is significantly less toxic to humanlung, liver, skin and eye cells than two commercial antimicrobialproducts currently on the market. Compared to some commercialchemicals that kill 100% human cells in vitro, AKWATON producesthe same effects as 70% ethanol, widely known and used all over theworld, killing only 30% of human cells. This study demonstrates thatAKWATON with all of these properties is an ideal antimicrobialproduct for hospitals, laboratories, food industries and householdfacilities.
AcknowledgementThe authors are grateful to the Research and Development
Committee of Université de Saint-Boniface for financial support, toFOSFATON-AKWATON INTERNATIONAL Ltd for providingAKWATON, and also to the reviewers for their guidance and helpfulcomments on the manuscript.
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Citation: Oulé MK, Lesage C, Gauvin J, Friesen M, Dickman M, et al. (2017) In Vitro Assessment of the Toxic Effects of an AKWATON based-disinfectant on Human Tissues. J Antimicrob Agents 3: 140. doi:10.4172/2472-1212.1000140
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